In general, the epsilon-amino group of lysyl residues is the most abundant positively charged moiety of protein structures. Besides being an important determinant of protein solubility and other protein environmental interactions, lysyl residues essential for catalytic activity have been suggested for a number of enzymes. Primarily on the basis of covalent modification studies and X-ray crystallographic structural analyses, essential lysyl residues have been implicated in (1) the binding of anionic substrates (e.g. ribonuclease), (2) the specificity of substrate binding (e.g. trypsin), (3) the binding between protein subunits (e.g. the nonactive site salt bridge between soybean trypsin inhibitor and trypsin), and (4) the binding of allosteric modifiers (e.g. heparin activation of antithrombin III). We propose the investigation of each of these postulated roles using covalent modification with small, amino group specific reagents which are enriched in 13C or 15N. Reductive methylation will be the primary mode of protein derivatization, though guanidination will also be developed as a means to introduce a reporter group. Nuclear magnetic resonance studies of enzymes derivatized with these spectroscopic probes offers a new dimension to this type of study.